Project Summary While RNA polymerase I (Pol I) activity is upregulated in many cancers, it remains unclear how to target the enzyme. Little is known about the stability of Pol I and the regulation of its activity. It is also unclear how to selectively target Pol I transcription in cancer cells without adversely affecting wild-type cells. We have recently discovered a first-in-class small molecule, BMH-21, that inhibits Pol I transcription and induces the degradation of RPA194, the catalytic subunit of Pol I. Treatment with this molecule results in cancer cell-specific cell death. Yet the mechanisms of this inducible degradation of RPA194 remain unknown. This project will investigate the mechanisms and regulation of RPA194 degradation and use the small molecule as a tool. Our prior data suggests that BMH-21 induces the ubiquitination of RPA194 and targets it for degradation by the proteasome. To test this premise and elucidate the mechanisms involved, this research training plan will pursue three primary goals. (1) To determine the site(s) of ubiquitination on RPA194; (2) To identify the E3 ligase(s) responsible for the ubiquitination of RPA194; (3) To elucidate the regulation of RPA194 degradation. These goals will be accomplished through the use of site-directed mutagenesis, ubiquitination analyses, and the identification of the ubiquitin chain linkages. E3 ligase candidates, identified in a previous screen, will be validated by knockdown and expression analyses, ubiquitination assays, and interaction analyses. Transcription assays, chromatin immunoprecipitation, and assays for Pol I complex assembly will be used to determine the regulation of RPA194 degradation. By studying the mechanisms and regulation of the turnover of RPA194, the study will provide fundamental new knowledge about the stability and regulation of Pol I. This knowledge will improve understanding of how to target Pol I as an anti-cancer therapeutic. The study will be conducted at the Johns Hopkins University School of Medicine, an institution which takes pride in the broad expertise of its faculty, numerous core facilities, and outstanding training environment. In addition to undertaking the proposed research, the trainee will take elective courses and participate in seminars to establish a strong foundation in molecular cancer biology. She will also collaborate with experts in ubiquitination and the regulation of Pol I transcription to enhance her training. She will present her research at internal retreats and external conferences, and she will write abstracts and manuscripts. Lastly, she will participate in workshops provided by the professional development office that cover topics such as interviewing, grant writing, and preparing a job talk. The fellowship will therefore have considerable impact in preparing the trainee for her career goal as an independent research scientist.